Manipulating the length of the b subunit F1 binding domain in F1F0 ATP synthase from Escherichia coli.

ABSTRACT

The peripheral stalk of F(1)F(0) ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F(0) to the top of the F(1) sector. The stalk serves as the stator necessary for holding F(1) against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F(1). Only the hydrophobic segment from val-121 to ala-132 and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F(1)F(0) complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F(1)F(0) ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F(1)F(0) complex by a normal b subunit.